Pseudomonas aeruginosa H181 and H185 are resistant to initial exposure to polymyxin B and continue to grow in its presence. Growth of the strains in the presence of 50 U of polymyxin B per ml was characterized by a doubling time of 120 min, whereas the doubling time in the absence of polymyxin was 60 min. Growth for two generations in the presence of polymyxin caused a 23 to 31% increase in lipopolysaccharide content. In addition, a marked increase in susceptibility to the detergents sodium deoxycholate, Triton X-100, and sodium dodecyl sulfate was observed. The resistant mutants had a small but significant reduction in their levels of dodecanoic acid as compared with the parent strain; however, this was the only consistent alteration observed in levels of fatty acids or readily extractable lipids. Polymyxin was fluorescently labeled by coupling to 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride). Growth of strains H181 and H185 in the presence of dansylated polymyxin resulted in a stable association between the fluorescent antibiotic and the outer membrane. We postulate that these alterations are part of an adaptive response by the strains to the presence of polymyxin in the growth medium and reflect a resistance mechanism distinct from the mechanism affording polymyxin B resistance when these strains are initially exposed to the antibiotic.